3.5Å cryoEM Structure of Hepatitis B Virus Core Assembled from Full-Length Core Protein

Abstract

The capsid shell of infectious hepatitis B virus (HBV) is composed of 240 copies of a single protein called HBV core antigen (HBc). An atomic model of a core assembled from truncated HBc was determined previously by X-ray crystallography. In an attempt to obtain atomic structural information of HBV core in a near native, non-crystalline environment, we reconstructed a 3.5Å-resolution structure of a recombinant core assembled from full-length HBc by cryo electron microscopy (cryoEM) and derived an atomic model. The structure shows that the 240 molecules of full-length HBc form a core with two layers. The outer layer, composed of the N-terminal assembly domain, is similar to the crystal structure of the truncated HBc, but has three differences. First, unlike the crystal structure, our cryoEM structure shows no disulfide bond between the Cys61 residues of the two subunits within the dimer building block, indicating such bond is not required for core formation. Second, our cryoEM structure reveals up to four more residues in the linker region (amino acids 140-149). Third, the loops in the cryoEM structures containing this linker region in subunits B and C are oriented differently (~30° and ~90°) from their counterparts in the crystal structure. The inner layer, composed of the C-terminal arginine-rich domain (ARD) and the ARD-bound RNAs, is partially-ordered and connected with the outer layer through linkers positioned around the two-fold axes. Weak densities emanate from the rims of positively charged channels through the icosahedral three-fold and local three-fold axes. We attribute these densities to the exposed portions of some ARDs, thus explaining ARD’s accessibility by proteases and antibodies. Our data supports a role of ARD in mediating communication between inside and outside of the core during HBV maturation and envelopment.

Nucleic Acid Chaperone Activity Associated with the Arginine-Rich Domain of Human Hepatitis B Virus Core Protein

“…2C). This prediction is consistent with the lack of structure at the C terminus of HBc in the context of an icosahedral particle as determined by cryo-electron microscopy (cryo-EM) (25,(34)(35)(36)(37) and X-ray crystallography (38).…”

“…One hallmark of nucleic acid chaperones is the lack of highly ordered structure (6,25). To assess the intrinsic disorder in the core protein of the HBV ayw (J02203) strain, we used the Protein DisOrder prediction program, developed by Ishida and Kinoshita (33) (http://prdos.hgc.jp/cgi-bin/top.cgi) (Fig.…”

“…Nucleic acid chaperone activity has been identified in nucleocapsid proteins of retroviruses, coronaviruses, hepatitis delta virus, hantavirus, and some Flaviviridae (20)(21)(22)(23)(24). Relative to the capsid proteins of many other viruses, HBc contains a very high helical content (25,26). To date, it remains unclear whether such a highly structured HBc protein could still contain any nucleic acid chaperone activity.…”

Distinct Viral Lineages from Fish and Amphibians Reveal the Complex Evolutionary History of Hepadnaviruses

“…The structures of the capsid dimer and homohexamer were based on the published structure of the HuHBV virion (Protein Data Bank [PDB] accession no. 3J2V and 5E0I, respectively) (32,33). Protein modeling and color manipulation were performed using PyMOL software (version 1.8.0.0) (http://www.pymol.org).…”

Core protein: A pleiotropic keystone in the HBV lifecycle

“…For T=4 capsids, there are two crystal structures and one cryo-EM structure at 4Å or better resolution (Bourne et al, 2006; Wynne et al, 1999; Yu et al, 2013). These provide a consistent picture where AB and CD dimers are nearly twofold symmetric.…”